Chronic infection with hepatitis C virus (HCV) is the primary cause of liver failure and hepatocellular carcinoma in many parts of the world; 170 million individuals worldwide are chronically infected with HCV. Current therapies for chronic HCV, such as systemic alpha-interferon, are expensive and applicable only to a minority of infected individuals. Chronic HCV therapy represents a huge unmet medical need. HCV has developed mechanisms to evade immune elimination, thereby allowing it to persist in the liver in the majority of infected individuals. We have defined an HCV consensus sequence for the most common HCV genotype in North America, Europe, Japan, and Australia and have shown that it is recognized by T-cells from individuals with divergent HCV sequences. The overall goal of this proposal is to select therapeutic vaccine candidates based on novel live-attenuated or Killed But Metabolically Active (KBMA) Listeria monocytogenes (Lm)-based platforms encoding hepatitis C virus (HCV) consensus sequence antigens that are poised for Phase l-enabling toxicology and immunogenicity studies in primates for eventual testing in humans with chronic HCV infection. We hypothesize that Lm will overcome the mechanisms of immune evasion by HCV. Lm induces a pro-inflammatory cytokine cascade, whose hallmark is the localized production of type I interferon by the liver, resulting in preferential accumulation and activation of natural killer (NK) and T cells in the organ. We hypothesize that the combination of HCV antigens based on the consensus sequence together with an Lm vaccine platform with an acceptable safety profile that naturally targets the adaptive and acquired immune response to the virus reservoir will result in an effective therapy for individuals with chronic HCV infection. Specifically, we propose to: (1) Construct Lm-HCV clinical candidate vaccine strains that encode the consensus HCV core, serine proteinase/helicase, and RNA dependent RNA polymerase proteins; (2) Select live-attenuated and KBMA Lm-HCV candidate vaccine strains for further studies in nonhuman primates based on presentation of characterized HLA A2-restricted antigens and activation of existing HCV-specific T cell lines, and on immunogenicity in HLA-A2 transgenic mice; (3) Develop fermentor-based process methods for manufacture of live-attenuated and KBMA Lm-HCV candidate vaccine strain lots for use in nonhuman primate studies; and, (4) Select a live-attenuated or KBMA Lm-HCV vaccine regimen for continued development based on immunogenicity and toxicity studies performed in baboons and a pilot therapeutic efficacy study in chronically infected chimpanzees. We have assembled an experienced consortium of scientists from academic and biotechnology institutions with expertise in novel vaccine platform development and translation, infectious disease vaccine development, study of HCV virology and immunology in both nonhuman primates and humans, and an existing productive collaboration.